Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

Abstract

BACKGROUND & AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the hYP7 and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NSG mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (controls), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NSG mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital PCR and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Authors

Li, Dan; Li, Nan; Zhang, Yi-Fan; Fu, Haiying; Feng, Mingqian; Schneider, Dina; Su, Ling; Wu, Xiaolin; Zhou, Jing; Mackay, Sean; Kramer, Josh; Duan, Zhijian; Yang, Hongjia; Kolluri, Aarti; Hummer, Alissa M; Torres, Madeline B; Zhu, Hu; Hall, Matthew; Luo, Xiaoling; Chen, Jinqiu; Wang, Qun; Abate-Daga, Daniel; Dropublic, Boro; Hewitt, Stephen M; Orentas, Rimas J; Greten, Tim F; Ho, Mitchell;

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